Fungi are a major source of clinical infections, especially among patients with compromised immune systems. The mechanisms fungi use to colonize human hosts are not fully understood, but are thought to often involve invasive growth. Specifically, many fungi are capable of attaching to and penetrating surfaces, such as those of human tissues. Saccharomyces cerevisiae, which is known to colonize immunocompromised humans, is a valuable model for understanding the environmental triggers and genetic mechanisms that underlie invasive growth in fungi. Although the S. cerevisiae reference strain S288C does not exhibit invasive growth, we have found that many ecologically and genetically diverse isolates can grow invasively, with expression of the trait often dependent on specific environmental conditions. In this proposal, (Aim 1) we use a combination of genetic mapping and genetic engineering to identify genes that cause variability in invasive growth among clinical isolates of S. cerevisiae. We conduct our genetic mapping studies in a panel of 2,880 segregants derived from the mating of 5 diverse strains in all 10 possible pairwise combinations. Once causal loci have been identified, we will use genetic engineering techniques to resolve these loci to specific genes and genetic variants. (Aim 2) We then test the potential relevance of the identified causal variants by infecting wild isolates and engineered strains into an animal model. We will inoculate the strains into a large number of wax moth larvae, which are commonly used to study the virulence of microbes, and measure the extent to which the strains cause sickness or death. Completion of the proposed research will provide detailed information about the genetic and environmental causes of invasive growth, and will also shed light on the potential clinical relevance of the identified alleles. Relevance: Cases of fungal pathogenesis are on the rise, with more than 500 species of fungi identified in human infections to date. Genetic approaches provide powerful tools for identifying the molecular mechanisms underlying pathogenesis in fungi. However, common opportunistic pathogens, such as Candida albicans, suffer from major limitations as genetic systems due to their inability to sexually reproduce in th lab. Isolates of S. cerevisiae exhibit substantial variability in the types of pathogenicity traitsthey exhibit, as well as the conditions in which these traits are expressed. To determine genetic and environmental factors that are involved in fungi attaching to and penetrating surfaces, which is thought to contribute to pathogenesis, we will perform genetic mapping experiments using multiple isolates of S. cerevisiae that were sampled from immunocompromised humans. The proposed research will improve general understanding of fungal pathogenesis and may identify potential targets for new antifungal drugs.